PROJECT SUMMARY/ABSTRACT Diabetic ketoacidosis (DKA) is a life-threatening condition that affects 200,000 patients annually and has a mortality rate of 4?5%. DKA pathology is the result of an insulin deficiency, which leads to an overproduction of ketone bodies in the form of acetoacetate (AcAc), ?-hydroxybutyrate (BHB), and acetone. Current strategies for the diagnosis of DKA rely on i) urine testing with nitroprusside paper strips for the detection of AcAc, and ii) blood testing with enzymatic electrochemical biosensors for the detection of BHB. Unfortunately, these tests are associated with well-known chemical limitations that lead to false-positive and false-negative results. Additionally, patients perceive urine and blood tests as unpleasant, time-consuming, invasive, and even distressing. This grant application will focus on the development of a noninvasive and patient-friendly enzyme-free electrochemical breathalyzer that chemoselectively analyzes acetone levels in the breath of patients with DKA. It is widely recognized that the small size of the ketone bodies generated during DKA enables them to be discharged into the endobronchial cavity to be exhaled in the form of volatile organic compounds (VOCs). Acetone, in particular, is a biomarker for DKA since it is the major VOC in the breath of patients at concentrations as high as 1250 ppm. In this proposal, we aim to replace enzymes traditionally used as biological recognition element in electrochemical biosensors with our click chemistry aminooxy (?ONH2) or hydrazine (?NHNH2) coatings, which are known to react rapidly, efficiently, and chemoselectively with carbonyl VOCs such as acetone. This project builds on the research strengths of the Laulh Group, which has extensive experience in selective derivatization of carbonyl metabolites, synthetic and electrochemical methodology, and an engineering background. We will test the hypothesis that acetone in breath can be readily trapped using aminooxy or hydrazine coatings and that the resulting adducts can be electrochemically oxidized at low oxidation potentials to obtain an electrochemical signal. Intensity and potential of the current generated through oxidation will provide qualitative and quantitative information about the levels of acetone in the breath sample. We propose the following 3 aims: Aim 1: Design and synthesize libraries of aminooxy- and hydrazine-containing coatings for carbonyl trapping. Aim 2: In vitro testing of coatings using acetone and potentiostat for electrochemical studies. Aim 3: Design a breathalyzer prototype. The primary impact of this proposal will be the proof-of-concept validation of an enzyme-free breathalyzer prototype for the diagnosis and management of diabetic ketoacidosis.